The gram-negative bacterium Vibrio cholerae causes cholera, a severe and sometimes lethal diarrheal disease. The genomes of Vibrio cholerae and related Vibrio species are distributed between two circular chromosomes, chromosome I (chrI) and chromosome II (chrII). Origins of replication of the two Vibrio cholerae chromosomes are undefined, and mechanisms regulating DNA replication in Vibrio cholerae were unknown.
The family Vibrionaceae, of which V. cholerae is the most clinically important member, includes several other human and fish pathogens such as Vibrio parahaemolyticus, Vibrio vulnificus, Photobacterium damselae, and Listonella anguillarum, and is one of the predominant families of marine microorganisms (Kita-Tsukamoto et al., 1993). The genomes of V. cholerae and several related Vibrio spp. are distributed between two circular chromosomes (Trucksis et al. 1998 and Yamaichi et al. 1999). This genomic structure was originally believed to be unusual among bacteria; it is now clear that many bacterial genomes—including those of several pathogens (e.g., DelVecchio et al., 2002)—consist of more than one chromosome.
Escherichia coli, which contains a single circular chromosome, has been the primary model organism used to elucidate mechanisms that control bacterial chromosome replication. Replication in E. coli initiates from a specific region of the chromosome, termed oriC. This 258 bp stretch of DNA is capable of autonomous replication and contains recognition sites for several replication factors (Messer et al., 1996). DnaA, the initiator protein, binds to 9 bp repeats within oriC, termed DnaA boxes (Fuller et al., 1984). This interaction stimulates DNA duplex separation at an adjacent region consisting of three AT-rich repeats, resulting in the formation of an open complex (Bramhill et al., 1988). DnaA is also believed to recruit a helicase, DnaB, to the open complex to fully unwind the strands (Marszalek et al., 1994). Once this prepriming complex is formed, RNA primers are synthesized and replication proceeds bidirectionally around the chromosome.
Initiation of replication in E. coli is a highly regulated event that occurs only once per cell cycle (Boye et al., 2000). Several mechanisms are thought to control initiation in E. coli. First, the methylation state of oriC regulates initiation (Boye et al. 1990 and Marinus 1996). oriC contains eleven sites for methylation by the enzyme DNA adenine methyltransferase (Dam). Ordinarily, E. coli DNA is fully methylated. However, newly replicated oriC is hemimethylated and becomes transiently unavailable for reinitiation because it is sequestered by SeqA, a protein that preferentially binds to hemimethylated DNA (Lu et al., 1994). Second, the availability of DnaA (amount of protein per cell) controls initiation, because it is titrated by binding to several high-affinity sites around the chromosome and thereby made unavailable for binding to oriC (Kitagawa et al., 1996). Third, the initiation potential is controlled by regulation of the activity of DnaA (Katayama et al., 1998).
Virtually nothing is known regarding replication control in prokaryotic organisms with multiple chromosomes. The genome of V. cholerae is distributed unequally between its two chromosomes; chromosome I (chrI) is larger than chromosome II (chrII) and contains most but not all of the genes essential for V. cholerae growth (Heidelberg et al., 2000). The presence of essential genes on chrII indicates that it is a bona fide chromosome as opposed to a dispensable plasmid. Bioinformatic analyses revealed that the putative origin of replication of chr I has sequence similarity to the origin of replication of the E. coli chromosome, oriC (Heidelberg et al., 2000). In contrast, the putative origin of replication of chr II lacks similarity to known origins and was assigned solely on the basis of GC nucleotide skew analysis (Heidelberg et al., 2000).